1) Field of the Invention
This invention relates to a novel process for synthesis of pyrrolo[2,1-c][1,4]benzodiazepine (PBD) analogues in a high yield. Specifically, this invention relates to a process for preparing PBD analogues starting from a substituted 2-amino benzoic acid, which enables a practical and large scale (e.g., ca. 10 g) synthesis of PBD analogues.
2) Description of the Related Art
Pyrrolo[2,1-c][1,4]benzodiazepines (PBDs) are a group of potent, naturally occurring anti-tumor antibiotics produced by Streptomyces species (M. D. Tendler et. al., Nature (1963), 199, 501; L. H. Hurley, J. Antibiot. (1977), 30, 349). The cytotoxic and antitumor effects of PBD compounds are believed to arise from their interaction with DNA molecules, which leads to inhibition of nucleic acid synthesis and production of excision-dependent single- and double-strand breaks in cellular DNA (K. W. Kohn, Anthramycin. In Antibiotics III Mechanism of Action of Antimicrobial and Antitumor Agents; ed. by J. W. Corcoran et. al. (Springer-Verlag, New York), pp. 3-11. (1975); R. L. Petrusek, et. al. J. Biol. Chem. 1982, 257, 6207). These antibiotics have been proposed to covalently bond to N2 of guanine to form a neutral minor groove adduct (L. H. Hurley et al., Nature (1979), 282, 529; S Cheatham et al., Med. Chem. (1988), 31, 583; J. J. Wang et al., Med. Chem. (1992), 35, 2995; J. A. Mountzouris et al., J. Med. Chem. (1994), 37, 3132).
Tomaymycin, cross-linker DSB-120 (J. A. Mountzouris et al., J. Med. Chem. (1994), 37, 3132; D. E. Thurston et al., J. Org. Chem. (1996), 61, 8141) and DC-81 (W. P. Hu et. al. J. Org. Chem. 2001, 66, 2881), the structure of which are shown below, are the best known examples of PBD analogues. 
Synthetic approaches of these PBD analogues have been reported (D. E. Thurston et. al. Chem. Rev. (1994), 94, 433 and references cited therein; A. Kamal et. al. Tetrahedron Lett. (2000), 41, 8631; T. Wang et. al. Org. Lett. (1999), 1, 1835.); however, most of them are tedious. For instance, the following scheme shows a widely used method which involves the cyclization of an amino dithioacetal (9) using mercuric chloride to yield an imine product. 
It is noted that it takes six steps to synthesize the starting material, i.e. (2S)-pyrrolidine-2-carboxaldehyde diethyl thioacetal, from L-proline. The overall yield of this 10-step synthesis process of DC-81 is about 15-20% (Thurston, D. E., et al., J. Org. Chem. (1996), 61, 8141. Thurston, D. E., et al., Synthesis (1990), 81). More recently, Wang et al. reported the total synthesis of DC-81 over 13 steps in 4% yield (Wang, T. et al., Org. Lett. (1999), 1, 1835).
Accordingly, there still exists a need to develop an efficient and practical process for the production of PBD analogues in a high yield.
Accordingly, in the first aspect, the present invention provides a process for preparing a compound of formula (I): 
wherein
R1 and R2 independently represent: hydrogen; halogen; amino; cyano; hydroxy; nitro; phenoxy; C1-C12 alkyl or C1-C12 alkoxy or C2-C12 alkenoxy optionally and independently substituted with halogen, amino, cyano, hydroxy, phenyl or C1-C3 alkoxy;
R3 represents: hydrogen, or alkyl or alkenyl or alkenylidene, or R form or S form of hydroxyl or alkoxy; and
R4 and R5 independently represent: hydrogen, halogen, cyano, hydroxy, phenoxy, C1-C6 alkyl or C1-C6 alkoxy optionally and independently substituted with halogen, amino, nitro, cyano, hydroxy, phenyl or C1-C3 alkoxy;
xe2x80x83the process comprising the steps of:
(a) reacting a substituted 2-amino benzoic acid compound of formula (II) with triphosgen to form an isatoic anhydride compound of formula (III): 
wherein
R1 and R2 independently represent: hydrogen; halogen; amino; cyano; nitro; phenoxy; C1-C12 alkyl or C1-C12 alkoxy or C2-C12 alkenoxy optionally and independently substituted with halogen, amino, cyano, hydroxy, phenyl or C1-C3 alkoxy; and
R4 and R5 independently represent: hydrogen, halogen, cyano, hydroxy, phenoxy, C1-C6 alkyl or C1-C6 alkoxy optionally and independently substituted with halogen, amino, nitro, cyano, hydroxy, phenyl or C1-C3 alkoxy; 
(b) coupling the isatoic anhydride compound of formula (III) from step (a) with an L-proline compound of formula (IV) to form a compound of formula (V): 
wherein R3 is hydrogen, hydroxyl, alkyl, alkenyl or alkenylidene or alkoxy; 
(c) converting the compound of formula (V) from step (b) to a compound of formula (VI) by reacting the compound of formula (V) with NaH, followed by reaction with methoxymethyl chloride (MOMCl): 
xe2x80x83and
(d) converting the compound of formula (VI) from step (c) to the compound of formula (I) by a reduction reaction in the presence of lithium borohydride (LiBH4); and
(e) when any one of R1, R2, R4 and R5 of the compound of formula (I) from step (d) is phenoxy or C1-C12 alkoxy substituted with phenyl, an optional step of converting said any one of R1, R2, R4 and R5 of formula (I) to a hydroxy group.
In a preferred embodiment, prior to step (a), the present process further includes an additional step of subjecting a substituted 2-nitrobenzoic acid of formula (IIA) to a reduction reaction to form the amine compound of formula (II): 
wherein R1, R2, R4 and R5 are the same as those defined for formula (II).
The reduction reaction of the additional step may be carried out: (1) by hydrogenation in the presence of a palladium-on-carbon system, (2) in the presence of an In/NH4Cl aqueous ethanol system, or (3) in the presence of a metal reducing agent selected from ferric chloride (FeCl3) and stannous chloride (SnCl2). In a more preferred embodiment, the reduction reaction of the additional step prior to step (a) of the present process is carried out in the presence of SnCl2.
In a further preferred embodiment, the optional step (e) of the present process is carried out in the presence of 1,4-cyclohexadiene.
In a preferred embodiment, the present process produces a compound of formula (I) wherein both R4 and R5 are hydrogen.
In a further preferred embodiment, the present process produces a compound of formula (I), wherein R1 and R2 independently represent halogen, cyano, phenoxy, hydroxy, or C1-C12 alkyl or C1-C12 alkoxy optionally and independently substituted with halogen, amino, cyano, hydroxy, C1-C3 alkoxy or phenyl. Preferably, R1 and R2 independently represent hydroxy, or C1-C12 alkyl or C1-C12 alkoxy optionally and independently substituted with halogen, amino, cyano, hydroxy, C1-C3 alkoxy or phenyl.
In a more preferred embodiment, the present process produces a compound of formula (I), wherein one of R1, R2, R4 and R5 is halo(en, cyano, phenoxy, hydroxy or C1-C12 alkyl or C1-C12 alkoxy or C2-C12 alkenoxy optionally substituted with halogen, amino, cyano, hydroxy, C1-C3 alkoxy or phenyl, and the others are hydrogen.
In a more preferred embodiment, the present process produces a compound of formula (I), wherein both R4 and R5 s are hydrogen, and R1 and R2 independently represent halogen, cyano, phenoxy, hydroxy, or C1-C12 alkyl or C1-C12 alkoxy or C2-C12 alkenoxy optionally and independently substituted with halogen, amino, cyano, hydroxy, C1-C3 alkoxy or phenyl. Preferably, R1 is benzyloxy and R2 is methoxy. In a further preferred embodiment, R1 is hydroxy and R2 is methoxy.
In a preferred embodiment, the present process produces a compound of formula (I), wherein R3 is hydrogen, ethylidene, or R3 is R form or S form of hydroxyl or alkoxy.
In a more preferred embodiment, the present invention provides a process for preparing 8-hydroxy-7-methoxy-pyrrolo[2,1-c][1,4] benzodiazepin-5-one, comprising the steps of:
(a)subjecting 4-benzyloxy-5-methoxy 2-nitrobenzoic acid to a reduction reaction to form 2-amino-4-benzyloxy-5-methoxybenzoic acid;
(b)reacting the 2-amino-4-benzyloxy-5-methoxybenzoic acid from step (a) with triphosgen to form 7-benzyloxy-6-methoxy-isatoic anhydride;
(c) coupling the 7-benzyloxy-6-methoxy-isatoic anhydride from step
(b) with L-proline to form 8-benzyloxy-7-methoxypyrrolo[2,1-c][1,4]benzodiazepine-5,11-dione;
(d) forming N-(10-methoxymethyl)-8-benzyloxy-7-methoxypyrrolo [2,1-c][1,4]benzodiazepine-5,11-dione by reacting the 8-benzyloxy-7-methoxypyrrolo[2,1-c][1,4]benzo-diazepine-5,11-dione from step (c) with NaH, followed by reaction with methoxymethyl chloride (MOMCl);
(e) converting the N-(10-methoxymethyl)-8-benzyloxy-7-methoxypyrrolo-[2,1-c][1,4]benzodiazepine-5,11-dione from step (d) to 8-benzyloxy-7-methoxy-pyrrolo[2,1-c][1,4]benzodiazepin-5-one via a reduction reaction in the presence of LiBH4; and
(f) reacting the 8-benzyloxy-7-methoxy-pyrrolo[2,1-c][1,4]benzo-diazepine 5-one from step (e) with 1,4-cyclohexadiene.
The above step (a) may be carried out in the presence of an In/NH4Cl aqueous ethanol system, or in the presence of a metal reducing agent selected from ferric chloride (FeCl3) and stannous chloride (SnCl2). In a preferred embodiment, the step (a) of the present process is carried out in the presence of SnCl2.
In a second aspect, the present invention provides a process for producing a compound of formula (I): 
wherein
R1 and R2 independently represent: hydrogen; halogen; amino; cyano; hydroxy; nitro; phenoxy; C1-C12 alkyl or C1-C12 alkoxy or C2-C12 alkenoxy optionally and independently substituted with halogen, amino, cyano, hydroxy, phenyl or C1-C3 alkoxy;
R3 represents: hydrogen, or alkyl or alkenyl or alkenylidene, or R form or S form of hydroxyl or alkoxy; and
R4 and R5 independently represent: hydrogen, halogen, cyano, hydroxy, phenoxy, C1-C6 alkyl or C1-C6 alkoxy optionally and independently substituted with halogen, amino, nitro, cyano, hydroxy, phenyl or C1-C3 alkoxy;
xe2x80x83the process comprising the step of subjecting the compound of formula (VI) to a reduction reaction in the presence of lithium borohydride (LiBH4): 
wherein
R1 and R2 independently represent: hydrogen; halogen; amino; cyano; nitro; phenoxy; C1-C12 alkyl or C1-C12 alkoxy or C2-C12 alkenoxy optionally and independently substituted with halogen, amino, cyano, hydroxy, phenyl or C1-C3 alkoxy; and
R3 is the same as that defined for formula (I);
R4 and R5 independently represent: hydrogen, halogen, cyano, hydroxy, phenoxy, C1-C6 alkyl or C1-C6 alkoxy optionally and independently substituted with halogen, amino, nitro, cyano, hydroxy, phenyl or C1-C3 alkoxy; and
xe2x80x83when any one of R1, R2, R4 and R5 of the resulting compound of (I) is phenoxy or C1-C12 alkoxy substituted with phenyl, an optional step of converting said any one of R1, R2, R4 and R5 of formula (I) to a hydroxy group.
This invention provides a very short route for efficient synthesis of PBD analogues, e.g. DC-81. The synthesis starts with the reaction of a substituted 2-amino benzoic acid with triphosgene in THF under reflux to form an isatoic anhydride compound, which is subsequently coupled with a substituted or unsubstituted L-proline compound in DMSO to produce a dilactam compound, followed by reaction with MOMCl. The resulting compound is then subjected to a reduction reaction in the presence of lithium borohydride (LiBH4).
More specifically, according to this invention, there is provided a process for preparing a compound of formula (I): 
wherein
R1 and R2 independently represent: hydrogen; halogen; amino; cyano; hydroxy; nitro; phenoxy; C1-C12 alkyl or C1-C12 alkoxy or C2-C12 alkenoxy optionally and independently substituted with halogen, amino, cyano, hydroxy, phenyl or C1-C3 alkoxy;
R3 represents: hydrogen, or alkyl or alkenyl or alkenylidene, or R form or S form of hydroxyl or alkoxy; and
R4 and R5 independently represent: hydrogen, halogen, cyano, hydroxy, phenoxy, C1-C6 alkyl or C1-C6 alkoxy optionally and independently substituted with halogen, amino, nitro, cyano, hydroxy, phenyl or C1-C6 alkoxy; the process comprising the steps of:
(a) reacting a substituted 2-amino benzoic acid compound of formula (II) with triphosgen to form an isatoic anhydride compound of formula (III): 
wherein
R1 and R2 independently represent: hydrogen; halogen; amino; cyano; nitro; phenoxy; C1-C12 alkyl or C1-C12 alkoxy or C2-C12 alkenoxy optionally and independently substituted with halogen, amino, cyano, hydroxy, phenyl or C1-C3 alkoxy; and
R4 and R5 independently represent; hydrogen, halogen, cyano, hydroxy, phenoxy, C1-C6 alkyl or C1-C6 alkoxy optionally and independently substituted with halogen, amino, nitro, cyano, hydroxy, phenyl or C1-C3 alkoxy; 
(b) coupling the isatoic anhydride compound of formula (III) from step (a) with an L-proline compound of formula (IV) to form a compound of formula (V): 
wherein R3 is hydrogen, hydroxyl, alkyl, alkenyl or alkenylidene or alkoxy; 
(c) converting the compound of formula (V) from step (b) to a compound of formula (VI) by reacting the compound of formula (V) with NaH, followed by reaction with methoxymethyl chloride (MOMCl): 
xe2x80x83and
(d) converting the compound of formula (VI) from step (c) to the compound of formula (I) by a reduction reaction in the presence of lithium borohydride (LiBH4); and
(e) when any one of R1, R2, R4 and R5 of the compound of formula (I) from step (d) is phenoxy or C1-C12 alkoxy substituted with phenyl, an optional step of converting said any one of R1, R2, R4 and R5 of formula (I) to a hydroxy group.
The suitable compound of formula (II) for use in step (a) of the present process may be prepared according to known methods with reference to, e.g., J. Org. Chem. USSR (1976), 12, 1045-1048; J. Chem. Soc. Commu. (1971) 567-572; Chem. Ber. (1913), 46, 3945; Tetrahedron (1967), 23, 4719; Chem. Ber. (1887), 20, 2441; Tetrahedron Lett. (1977), 3143; Eur. J. Med. Chem. Chim. Ther. (1999), 34 (9), 729-744 , Justus Liebigs Ann Chem. (1887), 237, 26; Am. Chem. J. (1889), 11, 7; and among others.
The suitable L-proline compound of formula (IV) for use in step (c) of the present process may be commercially available from, e.g. ACROS, or may be prepared according to known methods with reference to, e.g., J. Chem. Soc. (1965), 3850-3853; J. Chem. Soc. (1964), 5024-5029; Chem. Pharm. Bull. (1960) 8, 1110-1113; Chem. Ber. (1923) 56, 2214; Collet. Czech. Chem. Commu. (1995), 20 (1), 7; Acta Phys. Chem. (1957), 3, 118; Bull Chem. Soc. Jpn. (1981), 12, 3871-3872; J. Biol. Chem. (1952), 195, 383-384; J. Biol. Chem. (1953), 204, 307-313; Isr. J. Chem. (1974), 12, 165-166; Helv. Chim. Acta (1978), 61, 701-703; JMC (1967), 10, 1161-1162; Chem. Abstr., 66, 11176; Acta Chem. Scand. (1990) 44 (3), 243-251; Biochem. J. (1941), 35, 461-462; J. Biol. Chem. (1934), 595-599; JOC (1985) 50 (19), 3457-3462; JMC (1991) 34 (2), 717-725; Chem. Pharm. Bull. (1997) 45 (2), 255-259; Tetrahedron Letters (1991), 32 (26), 3049-3050; Tetrahedron Letters (1993), 34 (15), 2477-2480; J. Chem. Soc. Perkin Trans. (1995), 10, 1251-1258; JMC (1988), 31 (6), 1148-1160; Tetrahedron Letters (1986), 27 (2), 151-154; JOC, (1989), 54 (8), 1857-1866; Tetrahedron (1993), 49 (33), 7239-7250; JMC (1988), 31 (6), 1148-1160; JOC (1995), 60 (9), 2925-2930; JOC (1998), 63 (13), 4218-4227; J. Chem. Soc. Chem. Commu. (1987), 3, 166-168; Chemical Review (1994), 94 (2), pp. 454-455; and among others.
As an alternative, the substituted 2-amino benzoic acid of formula (II) used as a starting material of the present process may be obtained from a reduction of its corresponding substituted 2-nitrobenzoic acid (D. E. Thurston et al., Synthesis (1990), 81). Thus, prior to step (a), the present process may include an additional step of subjecting a substituted 2-nitrobenzoic acid of formula (IIA) to a reduction reaction to form the amine compound of formula (II): 
wherein R1, R2, R4 and R5 are the same as those defined for formula (II).
The reduction reaction of the additional step may be carried out: (1) by hydrogenation in the presence of a palladium-on-carbon system (K. C. Brown et al., Syn Comm. (1982), 12, 691), (2) in the presence of an In/NH4Cl aqueous ethanol system (C. J. Moody et al., Syn. Lett. (1998), 1028), or (3) in the presence of a metal reducing agent selected from ferric chloride (FeCl3) and stannous chloride (SnCl2). In a preferred embodiment, the reduction reaction of the additional step prior to step (a) of the present process is carried out in the presence of SnCl2.
When using a palladium-on-carbon system, the reduction reaction of the additional step may be conducted in a hydrogen atmosphere under a pressure of 2 ATM in the presence of 5% Pd/50% H2SO4 (aq.)/glacial acetic acid).
The suitable compound of formula (IIA) for use in this additional step may be prepared according to known methods with reference to, e.g., D. E. Thurston et al., Synthesis (1990), 81; J. Org. Chem. USSR (Engl. transl.) (1976), 12, 1057-1060; Tetrahedron, (1967), 23, 4719-4727; Acta Chem. Scand. (1948), 34, 35; Recl. Trav. Chim. Pays-Bas (1929), 48, 139; J. Med. Chem. (1991) 34 (3), 1142-1154; Chem. Pharm. Bull. (1996), 44 (5), 1074; Tetrahedron Letters (1995). 36 (35), pp.6333-6336; Tetrahedron (1997), 53 (9), pp. 3223-3230; J. K. Still et al., JACS (1989), 111, 5417; Bioorg. Med. Chem. Lett. (1997), 7 (14), 1875-1878; Eur. J. Med. Chem. Chim. (1999), 34 (9), 729-744; and among others.
It is found that a key step of the present synthesis process may reside in the reduction of the MOM-protected compound of formula (VI). Mori et al., reported that the imine form of PBD analogues could be prepared via reduction of an MOM-protected dilactam with 10 molar equiv of NaBH4 in MeOH at 0xc2x0 C., followed by silica gel chromatography (M. Mori et al., Tetrahedron Lett. (1985), 26, 5947). Unfortunately, as reported by Thurston et al. (Langley, D. R.; Thurston, D. E J. Org. Chem. (1987), 52, 91), after the MOM-protected dilactam was prepared, it failed to afford the corresponding imine compound a structure of formula (I), using either the conditions reported by Mori et al. (supra), or a number of variations. Instead, ring-opening products were obtained via 3-aza-Grob fragmentation (J. J. Wang et al., Tetrahedron (1998), 54, 13149).
In light of straightforward reaction sequence consideration, the applicants of this invention explored this step with different reagents for conversion of compound of formula (VI) to its imine form. After careful study, it was found that the MOM-protected dilactam compound of formula (VI) was successfully converted to the compound of formula (I) by treating with LiBH4 (1 molar equiv) in THF at xe2x88x9210xc2x0 C. for 9 h. Further attempts to complete the reaction with longer reaction time, more reagents, or higher temperature would produce over-reduction amine products.
When any one of R1, R2, R4 and R5 of the compound of formula (I) is phenoxy or C1-C12 alkoxy substituted with phenyl, the present process may include a further step of converting said any one of R1, R2, R4 and R5 of formula (I) to a hydroxy group according to known methods. For example, in the following Example 6, benzyl DC-81 was converted to DC-81.
Another advantage of the present process is that the reactions can be carried out at much larger scale (10 g) than previously reported syntheses. Furthermore, in the first two steps, as well as the additional step prior to step (a), of the present process, the products were easily recrystallized and pure enough for subsequent reactions. The intermediate compound of formula (III) can serve as a versatile leaping point for further analogue synthesis to establish the SAR of substituted prolidine C ring.
The following Examples are given for the purpose of illustration only and are not intended to limit the scope of the present invention.
In the following Examples, melting points are uncorrected. 1H NMR and 13C NMR spectra were recorded at 400 and 100 MHz, respectively, using CDCl3 as a solvent. 1H NMR chemical shifts are made with reference to TMS or CDCl3 (7.26 ppm). 13C NMR was made with reference to CDCl3 (77.0 ppm). Multiplicities were determined by the DEPT sequence as s, d, t, and q. Mass spectra and high-resolution mass spectra (HRMS) were measured using the electron-impact (EI, 70 eV) technique by Taichung Regional Instrument Center of the National Science Counsel (NSC) at National Chung-Hsing University (NCHU), Taiwan, ROC. Elemental analyses were performed by Tainan Regional Instrument Center of NSC at National Cheng-Kung University (NCKU), Taiwan, ROC. Flash chromatography was carried out on silica gel 60 (E. Merck, 230-400 mesh).